26
Review Article
Implications of glycoprotein VI for theranostics Meinrad Gawaz1; Sebastian Vogel1; Christina Pfannenberg2; Bernd Pichler3; Harald Langer1; Boris Bigalke1,4 1Department
of Cardiology & Cardiovascular Medicine, University of Tübingen, Germany; 2Department of Diagnostic and Interventional Radiology, University of Tübingen, Germany; 3Preclinical Imaging, University of Tübingen, Germany; 4King’s College London, Division of Imaging Sciences and Biomedical Engineering, London, UK
Summary Glycoprotein VI (GPVI), a membrane glycoprotein solely expressed in platelets and megakaryocytes, plays a critical role in thrombus formation due to collagen/GPVI-mediated platelet activation and adhesion. Recent studies have shown that surface expression of GPVI on circulating platelets is enhanced in acute cardiovascular diseases such as myocardial infarction and ischaemic stroke. Increased GPVI levels are associated with poor clinical outcome and are an early indicator for imminent myocardial infarction in patients with chest pain. The soluble form of the dimeric GPVI fusion protein (sGPVI-Fc) binds with high affinity to collagen and atherosclerotic plaque tissue. Non-invasCorrespondence to: Meinrad Gawaz, MD Department of Cardiology University of Tübingen Otfried-Müller-Straße 10 72076 Tübingen, Germany Tel.: +49 7071 29 83688, Fax: +49 7071 29 5749 E-mail: [email protected]
ive imaging studies with radiolabelled sGPVI-Fc show specific binding activity to vascular lesions in vivo. Further, sGPVI-Fc has been developed as a new therapeutic platelet-based strategy for lesion-directed antithrombotic therapy. This review summarises the potential of GPVI for diagnostic and therapeutic options based on novel non-invasive molecular imaging modalities to ameliorate care of patients with cardiovascular diseases.
Keywords Platelets, glycoprotein VI, biomarker, myocardial infarction, stroke, molecular imaging Financial support: Research in the authors’ laboratory is supported by the Deutsche Forschungsgemeinschaft (KFO 274 “Platelets – Basic Mechanisms and Translational Implications”). Received: September 13, 2013 Accepted after major revision: January 31, 2014 Epub ahead of print: February 20, 2014 http://dx.doi.org/10.1160/TH13-09-0756 Thromb Haemost 2014; 112: 26–31
Introduction Platelets are essential for primary haemostasis and thrombosis at sites of vascular injury (1, 2). In atherosclerotic lesions, extracellular matrix proteins are exposed towards the blood stream and propagate thrombosis (3). Fibrillar collagen is the major extracellular matrix protein in artery walls. Circulating platelets adhere to collagen, become activated and initiate thrombus formation. Glycoprotein VI (GPVI) is surface-expressed on platelets and is the prominent receptor that mediates platelet adhesion to collagen (4). GPVI is a type I transmembrane protein belonging to the immunoglobulin superfamily and is uniquely expressed in platelets and megakaryocytes (4–6). Up to 9,600 copy numbers of GPVI expressed per platelet have been estimated in a quantitative analysis of platelet protein (7). Whereas the dimeric form of GPVI binds with high affinity to collagen, the monomeric form does virtually binds with low affinity (8). Platelet activation by adenosine diphosphate (ADP) or thrombin receptor-activating peptide (TRAP) results in enhanced surface expression of GPVI on the plasma membrane and induces dimerisation of the receptor (9). Further, activated coagulation factor X (FXa) and high-shear forces may induce cleavage and shedding of the GPVI ectodomain dependent on metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family (10–12) leading to enhanced plasma concen-
trations of soluble GPVI. Thus, GPVI has raised great interest in cardiovascular science for its antithrombotic potential (13). Both direct inhibition of platelet-associated GPVI through specific antibodies as well as competitive inhibition of GPVI binding to immobilised collagen through soluble dimeric GPVI have been shown to inhibit arterial thrombosis with limited risk of bleeding in vivo (13-15). Due to the fact that GPVI is uniquely found on platelets and released as a soluble form into the plasma it has become an interesting diagnostic target for biomarker development, as well (16). Since GPVI-binding sites are primarily exposed on vulnerable vascular lesions to promote thrombus formation, soluble dimeric GPVI may be a convincing tool for molecular imaging of vulnerable plaques (17). Thus, the therapeutic and diagnostic (theranostic) potential of GPVI will make the receptor a promising target for the development of personalised strategies to improve efficacy of therapy in patients at high risk for atherothrombotic events such as myocardial infarction and ischaemic stroke.
GPVI as a thrombotic biomarker Platelet hyperaggregability and activation are one of the key mechanisms involved in the atherothrombotic complications associated
© Schattauer 2014
Thrombosis and Haemostasis 112.1/2014 Downloaded from www.thrombosis-online.com on 2014-11-07 | IP: 158.121.247.60 For personal or educational use only. No other uses without permission. All rights reserved.
27
Gawaz et al. GPVI and theranostics
with coronary and cerebrovascular diseases (18). Activation of circulating platelets is enhanced in acute coronary syndrome (ACS) and ischaemic stroke which is associated with poor outcome (19, 20). Low response to clopidogrel in patients with coronary artery disease treated with percutaneous coronary intervention (PCI) showed an increased risk for thrombo-ischaemic events and death (21). Moreover, cardiovascular risk factors such as diabetes mellitus are associated with an enhanced collagen-mediated platelet activation (22).
GPVI expressed on platelet-surface in patients with myocardial infarction and stroke Surface expression of GPVI on circulating platelets has been shown to be altered in cardiovascular diseases (23, 24) (▶ Table 1). The platelet Fc receptor that forms a functional complex with GPVI was significantly increased in patients with diabetes mellitus compared to those without diabetes. Fc receptor expression correlated with GPVI expression and was found to be independently associated with diabetes mellitus (25). In a consecutive cohort of 367 patients with symptomatic coronary artery disease, patients with ACS showed significantly enhanced GPVI expression on platelets compared with patients with stable coronary artery disease (23). In this study, the expression levels correlated with platelet degranulation markers such as CD62 and were independent of markers of myocardial necrosis such as troponin and creatine kinase. Moreover, patients with increased GPVI expression on hospital admission for acute chest pain had a 1.4-fold relative risk for ACS independent on myocardial necrosis marker troponin (▶ Figure 1) Table 1: GPVI as a biomarker for cardiovascular diseases.
(26). GPVI may thus be considered as an early marker for imminent acute coronary events in patients with chest pain (27). Further, platelet-associated GPVI was found to be enhanced in patients with ACS and ambiguous electrocardiogram (ECG) (28). High GPVI levels were also associated with increased residual platelet aggregation despite conventional dual antiplatelet therapy (26). In a large prospective study enrolling 2,213 consecutive patients who presented with chest pain, elevation of platelet GPVI was associated with a poor clinical outcome for composite events such as myocardial infarction, stroke, and cardiovascular death (29). Similar to ACS, patients with transient ischaemic attack (TIA) or ischaemic stroke showed enhanced expression of platelet GPVI (30). On patient hospital admission, enhanced GPVI levels were associated with a 2.4-fold relative risk for stroke and therefore with poorer clinical outcome in cumulative event-free survival for stroke, myocardial infarction, and cerebrovascular death at threemonth follow-up (▶ Figure 1). However, an increased affinity or avidity of the respective antibody used for detection of platelet GPVI following platelet activation might also contribute to the observed enhanced effects of GPVI expression. Conformational changes of GPVI such as receptor dimerisation have been shown to increase binding of platelets to collagen (9, 31). Thus, influence of such receptor modifications on binding of specific antibodies against platelet GPVI needs to be investigated in further studies.
Plasma levels of soluble GPVI (sGPVI) in cardiovascular diseases Upon platelet activation, GPVI is strongly expressed on the platelet surface and partially cleaved and shed from the plasma mem-
Marker
Usage
Target
Reference
pGPVI
diagnostic
AMI
(59)
pGPVI
diagnostic/prognostic
SAP/ACS
(23),(26)
sGPVI
diagnostic
Alzheimer’s Disease
(37)
pGPVI
diagnostic
Atrial Fibrillation/ACS
(24)
pGPVI
diagnostic
Platelet Count/ACS
(60)
pGPVI
diagnostic
Chest pain/ACS
(27)
pGPVI
diagnostic
Ambiguous ECG/AMI
(28)
pGPVI
diagnostic/prognostic
Stroke/TIA
(30)
pGPVI
prognostic
SAP/ACS
(29)
sGPVI
diagnostic
Stroke
(36)
sGPVI
diagnostic
DIC
(10)
sGPVI, pGPVI
diagnostic
SAP/ACS
(33)
sGPVI
therapeutic
ACS
(15)
sGPVI
diagnostic
Stroke
(61)
AMI – acute myocardial infarction; SAP – stable angina pectoris; ACS – acute coronary syndrome; ECG – electrocardiogram; TIA – transient ischemic attack; DIC – disseminated intravascular coagulation; pGPVI – platelet glycoprotein VI; sGPVI – soluble glycoprotein VI.
Thrombosis and Haemostasis 112.1/2014
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For personal or educational use only. No other uses without permission. All rights reserved.
28
Gawaz et al. GPVI and theranostics
Figure 1: Platelet surface expression of glycoprotein VI and survival at a three-month follow-up for patients with ACS and stroke. A) Patients with symptomatic coronary artery disease and elevated platelet collagen receptor glycoprotein VI (GPVI) expression (mean fluorescence intensity (MFI) ≥ 18.6) had a poorer clinical outcome in composite cumulative sur-
vival that included myocardial infarction, stroke, and cardiovascular death than patients with a decreased GPVI expression (log rank; p = 0.002) (modified according to (29)). B) These results were paralleled in composite cumulative survival in patients with ischaemic stroke at an MFI ≥ 18.6 (log rank; p = 0.045) (modified according to (30)).
brane as a soluble form of GPVI (sGPVI). Metalloproteinases ADAM 10 and ADAM 17 (11, 12), coagulation factor Xa (10), and high shear forces (32) have been documented to contribute to this cleavage and shedding process. Thus, increased levels of sGPVI can have different pathophysiological causes, and utilizing sGPVI as a biomarker for platelet-associated cardiovascular diseases might give us new insights for the diagnostic process. Recently, several sensitive detection reagents and assays for sGPVI have been developed (33). Elevated plasma levels of sGPVI have been described in patients with immune thrombocytopenia purpura (ITP) (34), lupus nephritis (35), dissiminated intravasal coagulation (DIC) (10), stable coronary artery disease (33), and acute ischaemic stroke (36). Decreased levels of sGPVI have been described in patients with Alzheimer disease (37) and with atrial fibrillation (24). Platelet activation is known to play a prominent pathophysiological role for disease progression and, thus, a plateletand plasma-based GPVI biomarker analysis may be a promising strategy to identify the state of cardiovascular diseases (acute vs stable). However, prospective and interventional studies are needed to further substantiate utility of GPVI for diagnostic purposes and risk managment.
(MI) or stroke. The majority of MIs are caused by vulnerable plaques with a lumen narrowing
Review Article
Implications of glycoprotein VI for theranostics Meinrad Gawaz1; Sebastian Vogel1; Christina Pfannenberg2; Bernd Pichler3; Harald Langer1; Boris Bigalke1,4 1Department
of Cardiology & Cardiovascular Medicine, University of Tübingen, Germany; 2Department of Diagnostic and Interventional Radiology, University of Tübingen, Germany; 3Preclinical Imaging, University of Tübingen, Germany; 4King’s College London, Division of Imaging Sciences and Biomedical Engineering, London, UK
Summary Glycoprotein VI (GPVI), a membrane glycoprotein solely expressed in platelets and megakaryocytes, plays a critical role in thrombus formation due to collagen/GPVI-mediated platelet activation and adhesion. Recent studies have shown that surface expression of GPVI on circulating platelets is enhanced in acute cardiovascular diseases such as myocardial infarction and ischaemic stroke. Increased GPVI levels are associated with poor clinical outcome and are an early indicator for imminent myocardial infarction in patients with chest pain. The soluble form of the dimeric GPVI fusion protein (sGPVI-Fc) binds with high affinity to collagen and atherosclerotic plaque tissue. Non-invasCorrespondence to: Meinrad Gawaz, MD Department of Cardiology University of Tübingen Otfried-Müller-Straße 10 72076 Tübingen, Germany Tel.: +49 7071 29 83688, Fax: +49 7071 29 5749 E-mail: [email protected]
ive imaging studies with radiolabelled sGPVI-Fc show specific binding activity to vascular lesions in vivo. Further, sGPVI-Fc has been developed as a new therapeutic platelet-based strategy for lesion-directed antithrombotic therapy. This review summarises the potential of GPVI for diagnostic and therapeutic options based on novel non-invasive molecular imaging modalities to ameliorate care of patients with cardiovascular diseases.
Keywords Platelets, glycoprotein VI, biomarker, myocardial infarction, stroke, molecular imaging Financial support: Research in the authors’ laboratory is supported by the Deutsche Forschungsgemeinschaft (KFO 274 “Platelets – Basic Mechanisms and Translational Implications”). Received: September 13, 2013 Accepted after major revision: January 31, 2014 Epub ahead of print: February 20, 2014 http://dx.doi.org/10.1160/TH13-09-0756 Thromb Haemost 2014; 112: 26–31
Introduction Platelets are essential for primary haemostasis and thrombosis at sites of vascular injury (1, 2). In atherosclerotic lesions, extracellular matrix proteins are exposed towards the blood stream and propagate thrombosis (3). Fibrillar collagen is the major extracellular matrix protein in artery walls. Circulating platelets adhere to collagen, become activated and initiate thrombus formation. Glycoprotein VI (GPVI) is surface-expressed on platelets and is the prominent receptor that mediates platelet adhesion to collagen (4). GPVI is a type I transmembrane protein belonging to the immunoglobulin superfamily and is uniquely expressed in platelets and megakaryocytes (4–6). Up to 9,600 copy numbers of GPVI expressed per platelet have been estimated in a quantitative analysis of platelet protein (7). Whereas the dimeric form of GPVI binds with high affinity to collagen, the monomeric form does virtually binds with low affinity (8). Platelet activation by adenosine diphosphate (ADP) or thrombin receptor-activating peptide (TRAP) results in enhanced surface expression of GPVI on the plasma membrane and induces dimerisation of the receptor (9). Further, activated coagulation factor X (FXa) and high-shear forces may induce cleavage and shedding of the GPVI ectodomain dependent on metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family (10–12) leading to enhanced plasma concen-
trations of soluble GPVI. Thus, GPVI has raised great interest in cardiovascular science for its antithrombotic potential (13). Both direct inhibition of platelet-associated GPVI through specific antibodies as well as competitive inhibition of GPVI binding to immobilised collagen through soluble dimeric GPVI have been shown to inhibit arterial thrombosis with limited risk of bleeding in vivo (13-15). Due to the fact that GPVI is uniquely found on platelets and released as a soluble form into the plasma it has become an interesting diagnostic target for biomarker development, as well (16). Since GPVI-binding sites are primarily exposed on vulnerable vascular lesions to promote thrombus formation, soluble dimeric GPVI may be a convincing tool for molecular imaging of vulnerable plaques (17). Thus, the therapeutic and diagnostic (theranostic) potential of GPVI will make the receptor a promising target for the development of personalised strategies to improve efficacy of therapy in patients at high risk for atherothrombotic events such as myocardial infarction and ischaemic stroke.
GPVI as a thrombotic biomarker Platelet hyperaggregability and activation are one of the key mechanisms involved in the atherothrombotic complications associated
© Schattauer 2014
Thrombosis and Haemostasis 112.1/2014 Downloaded from www.thrombosis-online.com on 2014-11-07 | IP: 158.121.247.60 For personal or educational use only. No other uses without permission. All rights reserved.
27
Gawaz et al. GPVI and theranostics
with coronary and cerebrovascular diseases (18). Activation of circulating platelets is enhanced in acute coronary syndrome (ACS) and ischaemic stroke which is associated with poor outcome (19, 20). Low response to clopidogrel in patients with coronary artery disease treated with percutaneous coronary intervention (PCI) showed an increased risk for thrombo-ischaemic events and death (21). Moreover, cardiovascular risk factors such as diabetes mellitus are associated with an enhanced collagen-mediated platelet activation (22).
GPVI expressed on platelet-surface in patients with myocardial infarction and stroke Surface expression of GPVI on circulating platelets has been shown to be altered in cardiovascular diseases (23, 24) (▶ Table 1). The platelet Fc receptor that forms a functional complex with GPVI was significantly increased in patients with diabetes mellitus compared to those without diabetes. Fc receptor expression correlated with GPVI expression and was found to be independently associated with diabetes mellitus (25). In a consecutive cohort of 367 patients with symptomatic coronary artery disease, patients with ACS showed significantly enhanced GPVI expression on platelets compared with patients with stable coronary artery disease (23). In this study, the expression levels correlated with platelet degranulation markers such as CD62 and were independent of markers of myocardial necrosis such as troponin and creatine kinase. Moreover, patients with increased GPVI expression on hospital admission for acute chest pain had a 1.4-fold relative risk for ACS independent on myocardial necrosis marker troponin (▶ Figure 1) Table 1: GPVI as a biomarker for cardiovascular diseases.
(26). GPVI may thus be considered as an early marker for imminent acute coronary events in patients with chest pain (27). Further, platelet-associated GPVI was found to be enhanced in patients with ACS and ambiguous electrocardiogram (ECG) (28). High GPVI levels were also associated with increased residual platelet aggregation despite conventional dual antiplatelet therapy (26). In a large prospective study enrolling 2,213 consecutive patients who presented with chest pain, elevation of platelet GPVI was associated with a poor clinical outcome for composite events such as myocardial infarction, stroke, and cardiovascular death (29). Similar to ACS, patients with transient ischaemic attack (TIA) or ischaemic stroke showed enhanced expression of platelet GPVI (30). On patient hospital admission, enhanced GPVI levels were associated with a 2.4-fold relative risk for stroke and therefore with poorer clinical outcome in cumulative event-free survival for stroke, myocardial infarction, and cerebrovascular death at threemonth follow-up (▶ Figure 1). However, an increased affinity or avidity of the respective antibody used for detection of platelet GPVI following platelet activation might also contribute to the observed enhanced effects of GPVI expression. Conformational changes of GPVI such as receptor dimerisation have been shown to increase binding of platelets to collagen (9, 31). Thus, influence of such receptor modifications on binding of specific antibodies against platelet GPVI needs to be investigated in further studies.
Plasma levels of soluble GPVI (sGPVI) in cardiovascular diseases Upon platelet activation, GPVI is strongly expressed on the platelet surface and partially cleaved and shed from the plasma mem-
Marker
Usage
Target
Reference
pGPVI
diagnostic
AMI
(59)
pGPVI
diagnostic/prognostic
SAP/ACS
(23),(26)
sGPVI
diagnostic
Alzheimer’s Disease
(37)
pGPVI
diagnostic
Atrial Fibrillation/ACS
(24)
pGPVI
diagnostic
Platelet Count/ACS
(60)
pGPVI
diagnostic
Chest pain/ACS
(27)
pGPVI
diagnostic
Ambiguous ECG/AMI
(28)
pGPVI
diagnostic/prognostic
Stroke/TIA
(30)
pGPVI
prognostic
SAP/ACS
(29)
sGPVI
diagnostic
Stroke
(36)
sGPVI
diagnostic
DIC
(10)
sGPVI, pGPVI
diagnostic
SAP/ACS
(33)
sGPVI
therapeutic
ACS
(15)
sGPVI
diagnostic
Stroke
(61)
AMI – acute myocardial infarction; SAP – stable angina pectoris; ACS – acute coronary syndrome; ECG – electrocardiogram; TIA – transient ischemic attack; DIC – disseminated intravascular coagulation; pGPVI – platelet glycoprotein VI; sGPVI – soluble glycoprotein VI.
Thrombosis and Haemostasis 112.1/2014
© Schattauer 2014 Downloaded from www.thrombosis-online.com on 2014-11-07 | IP: 158.121.247.60
For personal or educational use only. No other uses without permission. All rights reserved.
28
Gawaz et al. GPVI and theranostics
Figure 1: Platelet surface expression of glycoprotein VI and survival at a three-month follow-up for patients with ACS and stroke. A) Patients with symptomatic coronary artery disease and elevated platelet collagen receptor glycoprotein VI (GPVI) expression (mean fluorescence intensity (MFI) ≥ 18.6) had a poorer clinical outcome in composite cumulative sur-
vival that included myocardial infarction, stroke, and cardiovascular death than patients with a decreased GPVI expression (log rank; p = 0.002) (modified according to (29)). B) These results were paralleled in composite cumulative survival in patients with ischaemic stroke at an MFI ≥ 18.6 (log rank; p = 0.045) (modified according to (30)).
brane as a soluble form of GPVI (sGPVI). Metalloproteinases ADAM 10 and ADAM 17 (11, 12), coagulation factor Xa (10), and high shear forces (32) have been documented to contribute to this cleavage and shedding process. Thus, increased levels of sGPVI can have different pathophysiological causes, and utilizing sGPVI as a biomarker for platelet-associated cardiovascular diseases might give us new insights for the diagnostic process. Recently, several sensitive detection reagents and assays for sGPVI have been developed (33). Elevated plasma levels of sGPVI have been described in patients with immune thrombocytopenia purpura (ITP) (34), lupus nephritis (35), dissiminated intravasal coagulation (DIC) (10), stable coronary artery disease (33), and acute ischaemic stroke (36). Decreased levels of sGPVI have been described in patients with Alzheimer disease (37) and with atrial fibrillation (24). Platelet activation is known to play a prominent pathophysiological role for disease progression and, thus, a plateletand plasma-based GPVI biomarker analysis may be a promising strategy to identify the state of cardiovascular diseases (acute vs stable). However, prospective and interventional studies are needed to further substantiate utility of GPVI for diagnostic purposes and risk managment.
(MI) or stroke. The majority of MIs are caused by vulnerable plaques with a lumen narrowing
